Despite advances in modern chemotherapy, survival for patients with metastatic or relapsed Ewing sarcoma (ES) remains dismally low. In order to improve outcomes, it is necessary that we understand the molecular mechanisms that promote aggressive disease. It is our hypothesis that ES cells exhibit phenotypic plasticity, and that the phenotype can be influenced by selective pressures in the tumor microenvironment. To test this hypothesis, we have proposed two specific aims. First, we will assess the contribution of the extracellular ligand R-spondin (RSPO) to the metastatic phenotype. RSPO interacts with the surface receptor and stem cell marker LGR5. This interaction promotes canonical Wnt signaling. Based on our preliminary studies, we hypothesize that potentiation of Wnt signaling by RSPO will promote metastasis. In our second aim, we will elucidate the contribution of oncogenic EWS/ETS fusion proteins to the output of Wnt signaling in ES. EWS/ETS fusion proteins are the result of chromosomal translocations that occur in the majority of ES tumors. These fusion proteins interfere with Wnt signaling. We have observed that extracellular microenvironmental factors can alter EWS/ETS levels. It is our hypothesis that these changes in the microenvironment can alter EWS/ETS levels and thus contribute to regulation of Wnt activity. Through these studies, we expect to elucidate the specific role of RSPO/LGR5/Wnt signaling, and how this signaling is influenced by EWS/ETS fusion proteins. At the completion of these studies, we will have gained further insight into the molecular mechanisms that drive aggressive disease, and how these mechanisms can be exploited for therapy.